Mycoplasma is a eubacterium classified in the class Mollicutes. In a broad sense, not only the genus Mycoplasma but also the genus Ureaplasma, the genus Mesoplasma, the genus Entomoplasma, the genus Spiroplasma, the genus Acholeplasma, the genus Asteroleplasma, and the genus Thermoplasma are sometimes called Mycoplasma. Mycoplasma is the smallest self-replicable organism. At least 200 types of species are presently known. Mycoplasma has no peptidoglycan cell wall, which is commonly found in eubacteria. Because of this, the cell has an indefinite shape and flexibility. Since Mycoplasma has a small size of about 0.2 to 0.8 μm and indefinite in shape, the cell can pass through a sterilization filter of about 0.2 μm. Because of this, even if a cell culture medium is subjected to filter sterilization, Mycoplasma cannot be removed. In particular, certain species of Mycoplasma are known as representative bacteria causing microbial contamination in cell culture.
Due to the absence of a cell wall, Mycoplasma is not sensitive to antibiotics such as penicillin-based and cephem-based antibiotics, usually used in cell culture. Unlike other bacterial contaminants, Mycoplasma proliferates in a cell culture supernatant without causing visible changes such as a turbidity increase of medium and degeneration of cultured cell. Thus, Mycoplasma contamination is overlooked and spread unless Mycoplasma is found by the Mycoplasma detection method. Mycoplasma adsorbs to the cell membrane and depletes cell nutrients, thereby inhibiting cell proliferation and changing gene expression. For the reasons, experimental results from an infected culture are low in reliability. Accordingly, it is an important prerequisite to check the absence of Mycoplasma contamination before study. If cells infected with Mycoplasma are used for treatment in the field of regenerative medicine and cell therapy requiring cell culture, the cells negatively affect the immune system and may have a risk of causing pneumonia, urethritis and arthritis. Accordingly, it is essential to carry out a Mycoplasma test in production sites of biological material-derived medicines and clinical sites of regenerative medicine and cell therapy.
As the Mycoplasma test method, the Japanese pharmacopoeia proposes three methods, i.e., a culture method (agar and liquid medium method), a DNA staining method using indicator cells (indicator cell culture method) and a Nested PCR method, as reference information. However, the culture method has a problem in that a culture period of 28 days is too long. The DNA staining method has a problem in that the sensitivity is low. The Nested PCR method has a problem in that a false-positive due to carry over contamination of an amplified product is likely to occur. The three test methods are all insufficient as a safety test method for regenerative medicine and cell therapy used in practice. In the circumstances, a more practical method for detecting Mycoplasma has been in developing.
For example, Patent Document 1 discloses e.g., a primer pair for use in specifically amplifying a gene of Mycoplasma by real time PCR and a method for detecting Mycoplasma using the primer pair. In this method, 23S rRNA gene of Mycoplasma is used as an amplification target. Non-Patent Document 1 discloses e.g., a primer pair for use in specifically amplifying a gene of e.g., Mycoplasma, by real time PCR, a probe for detecting a product amplified by use of the primer pair and a method for detecting e.g., Mycoplasma by the primer pair and probe. In this method, tuf gene of e.g., Mycoplasma is used as an amplification target. Patent Document 2 discloses e.g., a primer set for use in specifically amplifying a Mycoplasma gene by a special gene amplification method called LAMP (loop-mediated isothermal amplification) method, a probe for detecting a product amplified by use of the primer set and a method for detecting Mycoplasma using the primer set and probe. In this method, 16S rRNA gene of Mycoplasma is used as an amplification target. Patent Document 3 discloses e.g., a kit containing a primer for use in specifically amplifying a gene of Mycoplasma by a real time nucleic acid amplification reaction (real time PCR) and a method for detecting Mycoplasma in a cell culture medium by use of the primer. In this method, rpoB gene of Mycoplasma is used as an amplification target. Likewise, a method for detecting Mycoplasma has been in developing. In the circumstance, a more practical Mycoplasma detection method by which a greater number of Mycoplasma species can be more quickly and easily detected with high sensitivity and accuracy has been desired.
Note that, Non-Patent Document 2 discloses that primers, which are used in a conventional Nested PCR method for detecting Mycoplasma, are used as primers for targeting 16S rRNA gene, 23S rRNA gene of Mycoplasma and the spacer region between both genes, for amplification. However, the conventional Nested PCR has a problem in that a false positive due to carry over contamination with an amplified product is likely to occur, as mentioned above.
In the meantime, certain species of Mycoplasma are known to cause pneumonia. For example, Patent Document 4 discloses e.g., a method for detecting a pneumococcus such as Mycoplasma pneumoniae by specifically amplifying a gene of the pneumococcus by e.g., PCR, for diagnosing pneumonia, a primer for use in the method and a complementary probe to a product amplified by the method. In this method, an amplification target for detecting Mycoplasma pneumoniae is DnaJ1 gene. As described, in order to diagnose pneumonia caused by Mycoplasma, development of a more practical detection method for Mycoplasma by which Mycoplasma can be more quickly and easily detected with high sensitivity and accuracy, has been desired.